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. 2012 Aug 16;7(8):e43532. doi: 10.1371/journal.pone.0043532

Figure 5. P2Y purinoceptor stimulation increases ARF6-GTP levels in human platelets.

Figure 5

(A and B) Human platelets were treated with ADP (10 µM; 0–5 min) and endogenous ARF-GTP levels assessed by using GST-GGA3 VHS-GAT pull down assay. Total ARF6 (A) or ARF1 (B) expression in platelet cell lysates and ARF6-GTP (A) or ARF1-GTP (B) precipitated with GST-GGA3 VHS-GAT beads were detected by immunoblotting using an anti-ARF6 mouse monoclonal and an anti-ARF1 rabbit polyclonal antibodies. The relative intensity of each ARF6-GTP or ARF1-GTP band was normalised to total ARF6 or ARF1 measured by densitometry (A and B). Values are mean ± SEM from three separate experiments. *p<0.05 compared with no ADP treatment alone control (Mann–Whitney U-test). (C) Human platelets were treated with thrombin (0.3 unit/ml) and collagen-related peptide (CRP; 5 µg/ml) for 0–5 mins and and endogenous ARF-GTP levels assessed. The data are representative of three independent experiments (D) Platelets were incubated with SecinH3 (15 µM) or vehicle alone for 30 minutes. Platelets were then treated with ADP (10 µM; 0–5 min) and endogenous ARF6-GTP levels assessed. The data represent mean ± SEM of three independent experiments. *p<0.05 compared with no ADP treatment alone control (Mann–Whitney U-test).